GFP Antibody (1025527) [Unconjugated]

Western blot shows synthetic eGFP, recombinant RANGAP-1 tagged with eYFP, recombinant GRPuv, and crystal jelly GFP. PVDF membrane was probed with 2 µg/mL of Mouse Anti-GFP Monoclonal Antibody (Catalog # MAB42402) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (HAF018). A specific band was detected for GFP at approximately 28 and recombinant RANGAP-1 tagged with eYFP at 45 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

GFP was detected in immersion fixed HEK293 human embryonic kidney cell line transfected with GFP (green; right panel) using Mouse Anti-GFP Monoclonal Antibody (Catalog # MAB42402) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red, left panel; NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. Staining was performed using our protocol for Fluorescent ICC Staining of Non-adherent Cells.

HEK293 human embryonic kidney cell line transfected with protein tagged with either eGFP (filled histogram) or mCherry (open histogram) was stained with Mouse Anti-GFP Monoclonal Antibody (Catalog # MAB42402) or Mouse IgG2B Isotype Control (MAB0041, data not shown) followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (F0101B). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (FC005). Staining was performed using our Staining Intracellular Molecules protocol.

Simple Western lane view shows eGFP and recombinant human RANGAP-1 tagged with eYFP, loaded at 0.2 mg/mL. Specific bands were detected for GFP at approximately 35 kDa and recombinant human RANGAP-1 tagged with eYFP at approximately 51 kDa (as indicated) using 10 µg/mL of Mouse Anti-GFP Monoclonal Antibody (Catalog # MAB42402) . This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

• Reactivity: All-Multi
• Applications: WB, Simple Western, Flow, ICC/IF
• Clone: 1025527
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated

GFP Antibody (1025527) [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant GFP
  aa 2-238
• Specificity: Detects GFP in direct ELISAs.
• Source: N/A
• Isotype: IgG2b
• Clonality: Monoclonal
• Host: Mouse
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry 8-25 ug/mL
  • Intracellular Staining by Flow Cytometry 0.25 ug/10^6 cells
  • Simple Western 10 ug/mL
  • Western Blot 2 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for GFP Antibody (1025527) [Unconjugated]

• eGFP
• Enhanced Green Fluorescent Protein
• GFP
• GFPuv
• green fluorescent protein (gfp)
• Green Fluorescent Protein

Background

Green fluorescent protein (GFP) is a 27 kDa protein originally isolated from the jellyfish victoria. In the presence of UV light (490-520 nm), it emits a green fluorescent color that can be used to pinpoint locations of various intracellular proteins. GFP is 238 amino acids (aa) in length. It is a globular monomer that has a tendency to dimerize. The monomer has the shape of a beta -barrel with a chromophore (aa 65-67) containing alpha -helix running up its center. +36 GFP is a superpositively charged GFP variant that can penetrate mammalian cells with potencies much greater than that of cationic peptides or modestly cationic engineered proteins.  Low molar concentrations of the protein can be observed within minutes of exposure.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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